Pyrrolidine alkaloids (e.g., nicotine) and tropane alkaloids (e.g., scopolamine and cocaine) are plant natural products that exhibit a diverse range of pharmacological activities. For example, nicotine may have utility for increasing cognitive function and is used in nicotine replacement therapy for smoking cessation. Tropane alkaloids are important anticholinergic drugs. Cocaine is used as a local anesthetic. These compounds are all isolated from plant sources for use as pharmaceutical drugs.
It would be of interest to enhance the production of pyrrolidine and tropane alkaloids in plants or plant cells by genetic engineering for the more efficient commercial production of these compounds for the pharmaceutical industry. Enhanced production of pyrrolidine and tropane alkaloids could be accomplished through selective breeding or genetic engineering using genes that encode enzymes in the biosynthetic pathways leading to these alkaloids. Genetic engineering for the accumulation of pathway intermediates, or reduction of end-product alkaloid levels, could be accomplished through classical or targeted mutagenesis approaches, such as Targeting Induced Local Lesions in Genomes (TILLING), or the specific silencing of genes encoding these enzymes via RNA interference and related techniques.
It would also be of interest to block the biosynthetic pathways leading to the production of these alkaloids at defined metabolic steps. The purpose of this would be to accumulate metabolic intermediates that may be of high value themselves or to generate plants that contain modified or reduced levels of end-product alkaloids. Tobacco plants that are genetically engineered to contain reduced nicotine levels may be useful for the production of plant-made pharmaceuticals, reduced-nicotine or nicotine-free cigarettes for use as smoking cessation aids (Benowitz et al. Clin Pharmocol Ther. 80(6):703-14 (2006)) and for use as low-toxicity industrial, food or biomass crops.
There are few genes known that encode enzymes involved in pyrrolidine or tropane alkaloid biosynthesis. This limits the ability to genetically engineer the pathways leading to these useful molecules.
An example of a known gene that encodes an enzyme involved in pyrrolidine alkaloid biosynthesis is the quinolate phosphoribosyl transferase (QPT) gene which has been cloned from N. tabacum and N. rustica; see U.S. Pat. No. 6,423,520 and No. 6,586,661, and Sinclair et al., Plant Mol. Biol. 44: 603-17 (2000). QPT suppression provides significant nicotinic alkaloid reductions in transgenic tobacco plants. Xie et al., Recent Advances in Tobacco Science 30: 17-37 (2004).
U.S. Pat. No. 5,369,023, and No. 5,260,205 discuss decreasing nicotine levels by suppressing putrescine N-methyltransferase (PMT) sequence. Suppression of an endogenous putrescine N-methyltransferase (PMT) sequence has been shown to reduce nicotine levels and increase anatabine levels by about 2-to-6-fold. Hibi et al., Plant Cell 6: 723-35 (1994); Sato et al. Proc Natl Acad Sci USA 98:367-72 (2001); Chintapakorn and Hamill, Plant Mol. Biol. 53:87-105 (2003); Steppuhn et al., PLoS Biol. 2:8:e217: 1074-1080 (2004). Overexpression of Nicotiana tabacum PMT in N. sylvestris resulted in an increase in nicotine content (Sato et al. Proc Natl Acad Sci USA 98:367-72 (2001)).
Suppression of endogenous A622 and NBB1 sequences has been shown to reduce nicotinic alkaloid levels in tobacco plants; see International patent publication WO 20061109197. A gene encoding a cytochrome P450 monooxygenase that converts nicotine to nornicotine has been cloned from N. tabacum. Siminszky B., et al., Proc Natl Acad Sci USA 102:14919-24, (2005); Gavilano et al. J. Agric. Food Chem. 54, 9071-9078 (2006).
The enzymatic activity of MPO was first detected in tobacco roots over three decades ago, Mizusaki S et al., Phytochemistry 11: 2757-2762 (1972), but the gene encoding this enzyme has remained unidentified until the present invention.
MPO plays a role in the pathway for the biosynthesis of alkaloids in plants, including medicinal tropane alkaloids. Hashimoto and Yamada, Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 257-285 (1994); Kutchan, In: Cordell, G. A. (ed.) ALKALOIDS (San Diego), vol. 50. Academic Press, Inc., San Diego, Calif., USA, (1998) pp. 257-316.
MPO enzymes have been purified from the roots of N. tabacum and N. rustica, Hyoscyamus niger, and Brugmansia candida x aurea hybrid and were shown to oxidize N-methylputrescine more efficiently than putrescine and cadaverine. Mizusaki et al., Phytochemistry 11: 2757-2762 (1972); Feth and Wagner, Phytochemistry 24: 1653-1655 (1985); Davies et al., Phytochemistry 28: 1573-1578 (1989); Hashimoto et al. Plant Physiol. 93:216-221 (1990); Walton and McLauchlan, Phytochemistry 29: 1455-1457 (1990); Haslam and Young, Phytochemistry 3 1: 40754079 (1992); McLauchlan et al., Planta 19: 440-445 (1993); Boswell et al., Phytochemistry 52: 871-878 (1999).
Anabasine and anatalline contain a piperidine moiety. The piperidine moiety of anabasine is thought to be derived from cadaverine via delta-1-piperidine in tobacco. Watson and Brown, J. Chem. Soc. Perkin Trans. 1: 2607-2610 (1990). Cadaverine is a good substrate for general diamine oxidases but is also a substrate for MPO, although it has a lower affinity than N-methylated diamines. Hashimoto et al. (1990), supra; Walton and McLauchlan, Phytochemistry 29: 1455-1457 (1990); Boswell et al., Phytochemistry 52: 871-878 (1999).
Katoh et al., Plant Cell Physiol. 48(3): 550-554 (2007), and Heim et al., Phytochemistry 68:454-463 (2007), both of which published after the filing of U.S. provisional application Ser. No. 60/814,542, disclose genes from tobacco that encode N-methylputrescine oxidase (MPO), which is involved in the nicotine biosynthetic pathway. There is no teaching of any method or use involving this gene for modifying nicotine production in plants, or for modifying production of any other alkaloid in plants.
Accordingly, there is a continuing need to identify additional genes whose expression can be regulated to decrease or increase the biosynthesis of alkaloids or to alter a plant's alkaloid profile by regulating the biosynthesis of a specific alkaloid(s).